Hatching agent for the potato cyst nematode

ABSTRACT

The invention is directed to a hatching agent for hatching the species and patho types of nematodes causing potation sickness, which nematodes comprise Globodera pallida, from the cysts thereof, with a molecular weight of 498, a component C 27  H 30  O 9  and the structure of formula (a) and the derivatives, esters and salts thereof. ##STR1##

The present invention relates to a hatching agent for the potato cystnematode, to a process for preparing such a hatching agent, and to amethod of combatting potato sickness.

Potato sickness, that is to say, the attack of the potato plant by thepotato cyst nematode (PCN) is a major problem. In particular for theproduction of industrial potatoes, which is often effected throughintensive potato culture, this attack results in a loss in production.In the culture of seed potatoes and consumption potatoes, such an attackalso occurs.

The potato plant acts as a host plant to the organisms causing potatosickness: nematodes, at present known by the names of Globoderarostochiensis and Globadera pallida, of which various pathotypes areknown. These nematodes bridge the time in which no potatoes are on thefield, i.e., from autumn until spring, or longer if there are no growingpotatoes or other host plants, in cysts freely occurring in the soil.These cysts are in fact the hardened abdomens, filled with eggs, offemale nematodes of a previous generation. In the spring, when thepotato plant is growing, the plant exudes hatching factors which lurethe larvae of the nematodes out of the eggs through the cyst wall to theplant.

Preventive measures and possible control of the nematodes have hithertosubstantially consisted of intensive crop rotation, measures of farmhygiene, the use of resistant varieties and soil disinfection.

As the financial yield per hectare for potatoes is more favourable thanfor other crops, intensive crop rotation is little attractive, and wherepossible, other measures are preferred.

Of the other measures, however, soil disinfection has been the only oneso far that found wide application. For this purpose 1,3-dichloropropeneand metam sodium are the main disinfectants used. In view of theconsiderable quantities of disinfectant used, and the chemical,physical, and toxicological properties of these agents, there is atendency of restricting the use of these agents. The most importantreason for it is that these agents are seen as a threat to theenvironment.

In the past, there has already been a considerable research for agentswhich artificially effect the hatching of the nematodes. In fact, if oneis capable of applying an agent to the soil while out of cultivation,which causes the PCN to hatch, one possibly has an effective biologicalmethod of controlling the organism. As, in fact, the nematodes have nosource of nutrition in such a situation, they will die, and thereafterpotatoes can be cultivated with less chance of damage from potatosickness. By combining such an agent with a small dose of a chemicalpesticide, a better effect could also be obtained.

Because, in the past, chemical control was effective, research into thebiological method was discontinued without any results being achieved.These investigations were reported, among other publications, inNematologica, 31, No. 2 (1985), pp 159-170. In it, the use of potatoroot diffusate for hatching the cysts is mentioned.

In the European patent application No. 434,417 a process for theproduction of a substance capable of stimulating the hatching of eggs ofpotato cyst nematodes from the root cells of plants of Solanaceae isdescribed.

It is an object of the present invention to provide a hatching agent forhatching the potato cyst nemerode. The invention relates to an agent forhatching the species and pathotypes of the nematodes causing potatosickness, which nematodes comprise Globodera rostochiensis and Globoderapallida, among others, from the cysts thereof, with a molecular weightof 498, a composition C₂₇ H₃₀ O₉ and the structure of the followingformula: ##STR2## and derivatives, esters and salts thereof.

After extensive and complicated research the above compound has beenidentified as the component responsible for the hatching of the potatocyst nematode. The systematic name of this compound istrans-2-(2,13-dihydroxy-9-methoxy-7,7,16-trimethyl-5,10,20-trioxo-19-oxahexacyclo[9.7.0.1³,6.0³,8.1¹²,15.0¹²,16]-eicosa-1(11),8-dien-15-yl)cyclopropanecarboxylic acid.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts the chemical structure, including the atomic numberingscheme, of the hatching agent compound.

The spectral data of the pure compound are given in the example.

The hatching agents are non-volatile compounds which are well-soluble inwater, methanol, diethyl ether, ethyl acetate and comparable polarsolvents, but are insoluble in hexane, dichloromethane, chloroform andthe like.

The hatching agent is unstable at pH below 2 and above 7 and attemperatures above about 35° C.

The hatching agent according to the invention can be isolated from ahydroculture, or from a tissue or cell culture of plant parts of membersof the nightshade family (Solanaceae), more specifically from potato ortomato roots, in the presence of a suitable nutrient source.

A suitable method of producing a hatching agent for the nematodesreferred to is production by means of a-hydroculture of planes of asuitable variety of the Solanaceae or a cell suspension culture of thetissues of such plants.

The hatching agent can also suitably be produced from the recirculatingfeed water of a substrate culture of members of the Solanaceae family,more in particular tomatoes. Surprisingly it has been found that therecirculating feed water of greenhouse substrate culture of tomatoes,usually on rockwool, contains substantial amounts of the hatching agent.

The hatching agent according to the invention can be obtained, forexample, using a cell suspension culture of the tissues of a suitablespecies variety of the Solaneacea. Using a suitable culture medium, forexample as described hereinafter, a solution of the hatching agent isobtained from such a cell suspension. The starting product may be thecell exudate, but it is also possible to use an extract or a homogenateof the cells proper. The cells are taken, for example, from tissues ofsuitable members of the nightshade family capable of hatching thesubject nematodes. Especially important are cultivated varieties of thespecies Solanum tuberosum L, for example, the Mentor variety. It ispossible to use the cells as such, or genetically modified cells, forexample, cells into which specific genetic information has beenintroduced using Agrobacterium tumefaciens. It is also possible to useshoot cultures, or root cultures, instead of cell suspension cultures.In these methods the same considerations apply with regard to the choiceof varieties as applied to the cell suspension cultures.

The media which may be suitable for the production of the hatching agentfrom a suspension of cells generally comprise:

basal components (salts, spore elements)

carbohydrate source (often sucrose, but it is also possible to use oneor more other carbohydrates)

vitamins (vitamin packets are commercially available as standardformulations, and can be used as such)

hormones (a combination often used is cytokinin/auxins, whether or notin combination with gibberellic acid and abscissic acid; however,composition and concentration can be varied depending on the type ofcell)

A possible combination of substrate components consists of:

Basal components+vitamins

According to Murshige and Skoog (Physiol. Plant 15, 473-497 (1962)).

Carbohydrate source sucrose solution, for example 30 g/l

Hormones

a. In the case of suspension cultures of non-genetically-modified cells,5 mg/l of naphthyl acetic acid may be added.

b. In the case of non-genetically-modified root cultures, 0.2 mg/lgibberellic acid and 0.05 mg/l naphthyl acetic acid may be added.

c. In the case of cultures on the basis of genetically transformed cells(cell suspension, root or shoot cultures) hormones need non always beused.

Naturally, the above substrate components are just examples ofcomponents which may be used, but it is by no means essential that thesevery components are used. The worker in the art may develop othercombinations and compositions resulting in a good hatching agentproduction on the basis of general knowledge in the art.

Depending on the nature of the culture, various processing proceduresmay be used to isolate the hatching agent from the culture medium orfrom the cells. A suitable procedure for isolating the hatching agentfrom the cells and the extracellular media may comprise the followingsteps:

1. homogenizing the total cell mass;

2. extraction or precipitation to remove proteins and cell debris;

3. if desired, filtering the liquid phase containing the hatching agent,and diluting or concentrating it to obtain the desired concentration;

4. purifying the liquid phase, using a preparative chromatographicpurification method.

Suitable chromatographic purification methods for recovering thehatching agent can be based upon one or more of the following propertiesof the hatching agent:

solubility in methanol

differences in the partition coefficient between water/methanol andwater/chloroform systems

charge in preparative/analytical chromatography using sepharose

hydrophobicity in reversed phase chromatography

charge/hydrophobicity ratio in ion exclusion chromatography

As indicated above, the hatching agent according to the invention is anon-volatile compound. Owing its low volatility, solutions containingthe agent can be effectively concentrated by means of vacuum techniques,such as by means of a rotary film evaporator, and/or freeze drying.

The hatching agent according to the invention can be obtained and usedin pure form, that is with a purity of at least 99 wt. %. However, foruse this may not be necessary and accordingly in can be obtained andused in partly purified form, for example such as can be obtained fromeluting an adsorbent on which the hatching agent has been adsorbed. Thisis especially useful in case the hatching agent is obtained from thecommercial tomato production on substrate in greenhouses. The feed waterused in the production, which is recirculated through the greenhousesand supplemented as necessary with fresh water, has been found tocontain substantial amounts of hatching agent. It is for examplepossible to recover the hatching agent continuously or discontinuouslyfrom the recirculating water, for example by adsorption or absorption.Elution of the hatching agent results in a concentrated hatching agentwith an aqueous solution as carrier.

The hatching agent can be used as such, that is in the acid form, assalt, for example the sodium or potassium salt, as ester with a suitablecompound, or as a derivative, a substituted compound and the like.

The hatching agent can be introduced into the soil as such, or in theform of a preparation with a suitable carrier, such as water or anaqueous solution. A solid carrier can also be used.

To obtain a prolonged activity the use of a controlled releaseformulation can have advantages. Suitable controlled releaseformulations are well-known, for example such as can be used forcomponents to be used in soil.

Hatching agents in purified or partly purified form are active inaqueous solutions, or as a solid (optionally in formulations) in aconcentration ranging from 0.01 to 1000 mg/kg soil, with the optimumranging from 1 to 100 mg/kg soil.

For best results, the solution or solid, optionally in a formulation,should be introduced or injected into the soil at a depth of preferablyabout 10-20 cm. On fenny soils, more of the larvae of the PCN arehatched than on other soils, and also significantly more than byspontaneous hatching.

Surprisingly, it has been found that the activity of the hatching agentaccording to the invention, both in the pure form and in the form of anunpurified preparation from a hydroculture, cell culture or a tissueculture, can be considerably enhanced by using it in an acidiccomposition, for example in combination with ascorbic acid.

Hatching agent preparations can be tested for activity with reference tothe hatching of larvae from eggs.

For this purpose, cysts are cleaned of root fragments and/or dirt andsieved over 0.5 mm and 0.25 mm sieves. 32 mg of the fraction >0.5 and22.5 mg of the 0.25-0.5 fraction have been weighed: Together thisamounts to about 1200 cysts. The solid to be tested is dissolved in tapwater to a concentration equivalent to 125 mg/l of the original startingmaterial. Of this stock solution, a 10-fold and a 100-fold dilution aremade.

The egg suspension is incorporated in 70 ml tap water and transferred toa graded cylinder and insufflated with air by means of an aquarium pump.

5 ml hatching agent solution and 0.5 ml egg suspension are pipetted intopolystyrene tubes and then sealed from the air. As a control, 0.5 ml eggsuspension is pipetted into 5 ml tap water. Thereafter the tubes arestored with rotation at 20° C.

Eggs and remaining nematode worms are counted at t=0 days and worms arecounted at t=4, 5 and/or 6 days. For this purpose, the tube is firstvigorously shaken manually for 4 seconds and then with a Vortex mixerfor another 10 seconds. When the suspension has reached quiescence, 0.5ml is pipetted out of the tube at a level of 1 cm below the liquidsurface. This sample is placed on a counting dish and diluted with freshtap water until the water film covers the entire bottom. The eggs andworms are counted under a binocular.

The calculations were performed as follows: Absolute activity: ##EQU1##

The reaction of the free eggs to the presence of hatching agent can bedescribed using the Monod kinetics for the growth of micro-organismswith a non-competitive inhibition of a substrate.

EXAMPLE

About 700 potato plants (of the Mentor variety) were cultured inhydrocultures using a recirculating feed solution containing potassiumnitrate, calcium nitrate, potassium dihydrogen phosphate, ammoniumsulfate, magnesium sulfate, iron (II) sulfate, mangane (II) sulfate,zinc sulfate, copper (II) sulfate and sodium borate. The phosphate andnitrate contents of the feed solution is measured and, if necessary,adjusted. The pH is kept at a value below 4.2 by adding a solution of 6%nitric acid and 4.25% phosphoric acid, if necessary.

By passing the recirculating feed solution through a column containingAmberlite XAD-2, hatching agent was absorbed on the column.

When changing the feed solution the XAD-2 column is removed and washedwith distilled water until the conductivity is below 7×10⁻⁶ S. Theadsorbed material was desorbed from XAD-2 by successively eluting with60% methanol-water and methanol. The eluate was concentrated and driedby means of a rotary vacuum evaporator and freeze drier.

This hatching agent preparation was subjected to a number ofpurification steps based upon

differences of solubility in methanol and water

partition coefficient in counter-current extraction

charge in preparative/analytical chromatography using sepharose

hydrophobicity in reversed phase chromatography

charge/hydrophobicity ratio in ion exclusion chromatography

For this purpose the preparation was dissolved in methanol and shakenuntil a homogeneous mixture had formed. After centrifugation, the clearsupernatant was removed by means of a pipette, and concentrated anddried by means of a rotary vacuum evaporator and freeze drier.

The piping system of a Droplet Counter Current Chromatograph (DCCC) wasfilled with the top layer of a mixture of chloroform, methanol and water(35:65:40). The hatching agent preparation was dissolved in the toplayer of the mixture and introduced into the column. The hatching agentswere eluted with the bottom layer of the mixture referred to andcollected in fractions. The stationary phase was pumped out of thecolumn, and also collected in fractions. The acivity of the collectedfractions of the mobile and stationary phases were tested separately.

Thereafter the fractions were fractionated over an anion exchanger, e.g.Q Sepharose HP. For this purpose, the hatching agent was dissolved andintroduced into the column in a buffer (e.g. 5 mM piperazine/acetic acidpH 6.0) and eluted by means of a gradient with another buffer (forexample 5 mM piperazine+1M sodium acetate pH 6.0) after the non-bondedmaterial had left the column. The eluate was collected in fractions andtested.

The combined active fractions of the anion exchanger were fractionatedover a preparative Rsil C18 column (250×25 mm) by means of gradientelution. The active fractions were combined, concentrated andfreeze-dried. This material was dissolved in 1 mM HCl and fractionatedover a HPX-87H column. The active fractions were combined and elutedover two series connected Bakerbond C18 columns. In this way 245 μg purehatching agent was obtained, which crystalized from a supersaturatedsolution. The hatching agent thus obtained was subjected to variousanalyses in order to determine the chemical structure thereof. Thisresulted in the following structure for the hatching agent: ##STR3##

This hatching agent had a K_(m) value, as defined in accordance with theMonod kinetics of about 10⁻⁸ g/l. Based upon this value the hatchingagent content of the unpurified solutions can be determined using theequation: ##EQU2## in which K_(m) is the concentration value at whichhalf of the maximum hatching by the unpurified agent has been found.

Spectral analyses of hatching agent Mass spectrometry

The analyses of purified hatching agent were performed with FinniganMAT-95Q, Finnigan MAT TSQ 700 MS/MS and Finnigan MAT Laser TOF massspectrometers.

The mass-spectrum of the material in acid form yielded the molucule ionhaving a molecular weight of 498 and a number of fragments withmass/charge of 480, 470 and 450 Dalton. The electrospray and laserdesorption mass-spectrum of the sodium salt gave a mass/charge of 521Dalton.

Proton NMR

The hatching agent was dissolved in 0.5 ml D₂ O and placed in aVarian-400 spectrometer. The scanning time was about 15 hours (4720transients). A spectrum was also made from a comparable control.

In the proton NMR spectrum, the following shifts and couplings werefound, using a Varian-Unity 400 MHz spectrometer in D₂ O at 5° C.,external reference sodium salt of 3-(methylsilyl)-propionic acid-d₄, 0.0ppm. The sample was used as the sodium salt:

    ______________________________________                                        Carbon    Shift      Couplings                                                position  (ppm)      (Hz)                                                     ______________________________________                                        2             4,09       --                                                   4             2,61       17,90 (4')                                           4'            2,61                                                            6             4,04       --                                                   7     (Me)    1,20       --                                                                 1,21       --                                                   9     (OME)   3,26       --                                                   13            4,32       7,68 (14) 2,74 (14')                                 14            2,18       7,54 (13) 12,47 (14')                                14'           2,04       12,60 (14) 3,02 (13)                                 16    (Me)    1,34       --                                                   17            1,95       4,94 (18') 14,47 (17')                               17'           1,43       5,21 (18) 13,57 (17') + ? (18')                      18            2,48       5,21 (17') 20,43 (18')                               18'           2,58       pattern unclear                                      Cyclopropane moiety                                                           1             1,78       multiplet                                            2             1,50       multiplet                                            3             0,85       doublet                                              3'            0,84       dubbel doublet                                       ______________________________________                                    

Infrared analysis

Pure hatching agent was dissolved in methanol and transferred to a KBrfilled microcup. After careful evaporation the spectrum was determined.As blanc pure methanol was prepared in the same manner. The spectra havebeen determined using a Bruker IFS-85 FTIR spectrometer, DTGS detector,optical resolution 4 cm⁻¹, number of scans 128/256. The spectrum wastransformed using the Kubella-Munk transformation. The absorption bandscould be assigned as follows:

    ______________________________________                                        Wavenumber (cm.sup.-1)                                                                          Functional group                                            ______________________________________                                        3379              --OH, including H.sub.2 O                                   2949/1470         --CH.sub.3 and/or CH.sub.2 in 5-ring                        2838              --OCH.sub.3                                                 1765              >C═O in 4-ring                                          1660              C═C                                                     Complex bonds in 1200-100                                                                       Various aliphatic ether-                                                      oxygen atoms                                                ______________________________________                                    

UV

The UV spectrum of the hatching agent is characterized by an absorptionmaximum at 267 nm and one at about 200 nm. The ratio of the extinctionsat 225 nm and 267 nm is about 1.5 for the pure hatching agent. Theextinction coefficient at 267 nm is 4550±1250 l.mol⁻¹. This absorptionsuggests the presence of a conjugated system of C═C and/or C═O bonds,which corresponds with the structure of the hatching agent of theinvention.

Rontgen diffraction analysis

The crystal was measured on an Enraf-Nonius CAD-4 diffractometer withgraphite-monochromated CuKα radiation and α-2θscan. A total of 10551reflections was measured within the range -13≦h≦13, -25≦k≦0, -14≦l≦14,the unique set consits of 5257 reflections. Of these, 3721 were abovethe significance level of 2.5 σ(I). The maximum value of (sinθ)/λ was0.61 Å⁻¹. Two reference reflections (021,200) were measured hourly andshowed 9% decrease during the 116 h collecting time, which was correctedfor. Unit-cell parameters were refined by a least-squares fittingprocedure using 23 reflections with 74<2θ<84°. Corrections for Lorentzand polarisation effects were applied. The structure was solved by theprogram CRUNCH, which uses Karle-Hauptman matrices to determine thephases. The water molecules were found in a subsequent ΔF synthesis.Anisotropic block-diagonal least-squares refinement on F, converged toR=0.126, R_(w) =0.171, (Δ/σ)_(max) =0.85. The goodness of fit S=0.548. Aweighting scheme w=(3.0+F_(obs) +0.011*F_(obs) ²)⁻¹ was used. Anempirical absorption correction (DIFABS, Walker and Stuart 1983) wasapplied, with coefficients in the range of 0.23-1.43. Scattering factorswere taken from Cromer and Mann (1968); International Tables for X-rayCrystallography (1974). All calculations were performed with XTAL (Halland Stewart 1990), unless stated otherwise. The atomic numbering schemeis shown in FIG. 1.

We claim:
 1. A hatching agent composition consisting essentially of acompound of the following formula: ##STR4## or a salt or an ester of thecompound as the active ingredient to effect hatching of potato cystnematodes,said active ingredient having been isolated from therecirculating feed water of a culture of a plant of the familySolanaceae.
 2. The composition of claim 1 wherein said active ingredientis isolated from said recirculating feed water by adsorption of theactive ingredient on a chromatographic column and thereafter eluting theactive ingredient from said column.
 3. The composition of claim 2wherein the active ingredient is eluted from the column with an aqueoussolution.
 4. The composition of claim 1 in combination with a carrier.5. The composition of claim 4 wherein said carrier is water or anaqueous solution.
 6. The composition of claim 4 wherein said carrier isa solid.
 7. The composition of claim 1 wherein the culture is oftomatoes or potatoes.
 8. A hatching agent composition consistingessentially of a compound of the following formula: ##STR5## or a saltor an ester of the compound as the active ingredient to effect hatchingof potato cyst nematodes,said active ingredient having been isolatedfrom the recirculating feed water of a culture of a plant of the familySolanaceae and wherein said active ingredient has a purity of at least99 wt. %.
 9. A hatching agent composition consisting essentially of acompound of the following formula: ##STR6## or a salt or an ester of thecompound as the active ingredient to effect hatching of potato cystnematodes, said active ingredient having been isolated from therecirculating feed water of a culture of a plant of the familySolanaceae, in combination with soil disinfectants selected from thegroup consisting of metam sodium and 1,3-dichloropropene.